Prokaryotic expression of Campylobacter jejuni mcp1/2/3 genes and relationship among the expressed products and bacterial chemotactic behavior / 中华微生物学和免疫学杂志

ABSTRACT

Objective To clone mcp1, mcp2 and mop3 genes that encoding methyl-accepting chemotaxis proteins(MCP) of Campy/obacter jejuni for construction of their prokaryotic expression systems, and to establish chemotactic model in vitro of the microbe for determining chemotaxis-inducing substances and to understand relationship among MCPs and chemotactic inducers. Methods The segments of mep1, mcp2 and mcp3 genes were amplified by PCR and then sequenced after T-A cloning. Prokaryofic expression systems of the genes were subsequently constructed. SDS-PAGE pins Bin-Rod Gel Image Analyzer were used to examine the expression of target recombinant proteins rMCP1, rMCP2 and rMCP3, and Ni-NTA affinity chromatography was performed to purify the rMCPs. Rabbits were immunized with each of the three rMCPs to obtain antisera. Immunodiffusion assay was performed to measure the titers of antisera. IgG in each of the antisera were extracted by ammonium sulfate precipitation and DEAE-32 ion exchange chromatography, and IgG F(ab')2 were then prepared by pepsin enzymolysis and Sephadex G-100 chromatography. Chemotactic model in vitro of C. jejuni based on HAP( hard-agar plus) method was established to determine the chemo-taxis-inducing effect of eight candidate substances. Chemotaxis inhibition test based on IgG F(ab')2 bloc-king was applied to determine the function and diversity of MCPs. Results The segments with expected si-zes amplified from mcp1, racp2 and mcp3 genes were obtained by PCRs, and their nucleotide and putative amino acid sequences were completely same as the reported. The constructed prokaryotic systems could effi-ciently express rMCPs with the yields about 10% of the total bacterial proteins. Immunization with rMCP1, MCP2 and rMCP3 enables the rabbits to produce specific antibody. All the antisera had 1 4 immunodiffusion titers. Both bovine bile and sodium deoxycholate(DOC) were able to induce ehemotactie movement of C. je-juni in a dosage-dependent manner ( P < 0.05 ). When MCP1 and MCP2 were blocked with their IgG F(ab')2, the ehemotaetic ability of C. jejurd were remarkably decreased (P <0. 05). However, MCP3 blocking did not affect the chemotaxis ( P > 0.05 ). Conclusion The C. jejuni MCPs are successfully ex-pressed in this study. Bovine bile and DOC are the inducers for chemotaxis of C. jejuni, and MCP1 and MCP2 are involved in the process of ehemotaxis iadueed by DOC.